Specificity comparison using ELISA

The antibodies were titrated against a single-stranded DNA oligo containing either a single unmodified cytosine or differentially modified cytosines: 5-mC, 5-hmC, 5-caC, and 5-fC.

The graphs above demonstrate that the 5-mC rabbit mAb from CST (left) binds to the oligo containing methylated cytosine with great specificity, showing negligible binding to other cytosine modifications, whereas the 5-mC mouse mAb (clone 33D3; right) shows significant cross-reactivity to other forms of modified cytosine, most prominently 5-fC and 5-hmC and, to a lesser extent, 5-caC. In addition clone 33D3 also exhibits considerable cross-reactivity to unmethylated DNA.

As shown in the graphs above the 5-fC rabbit mAb from CST (left) binds strictly to the oligo containing formylcytosine, whereas a well known 5-fC polyclonal antibody from another company (right) shows significant cross-reactivity to 5-hmC and, to a lesser extent, other methylation variants.

Specificity comparison using DNA IP

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 was further tested by performing DNA IPs and compared to that of clone 33D3. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with control DNA containing either unmethylated cytosine, 5-mC, or 5-hmC. IPs were performed using 5-mC (D3S2Z) Rabbit mAb and the SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. The enriched DNA was quantified by real-time PCR using primers specific to the spiked-in control DNA sequence. The amount of immunoprecipitated DNA in each sample is represented as percent of total amount of input DNA.

The bar graph clearly indicates that the 5-mC rabbit mAb from CST shows select reactivity to methylcytosine with no binding to hydroxymethylcytosine. In contrast clone 33D3 shows dramatic cross-reactivity to hydroxymethylcytosine with only modest reactivity to methylated cytosine, clearly highlighting the promiscuity of this antibody.